Processes and compositions for production of Anti-CD19 antibodies

Abstract

The invention relates to the methods of treatment of diseases by administration of monoclonal anti-CD19 antibodies prepared in Chinese Hamster Ovary (CHO) cells.

Description

[0001] Patent Application CLIMB -009 / 01 WO 40193 / 34

[0002] PROCESSES AND COMPOSITIONS FOR PRODUCTION OF ANTI-CD19 ANTIBODIES

[0003] I. Field of the Invention

[0004] The invention relates to the methods of treatment of diseases by administration of monoclonal anti-CD19 antibodies prepared in Chinese Hamster Ovary (CHO) cells.

[0005] II. Sequence Listing

[0006] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named CLIMB-009-0 IWO-Seq-Listing. xml, created on October 10, 2025, which is 55 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0007] III. Background

[0008] CD 19 is a surface protein found on B cells and on certain cancerous cells derived from B cells, such as many B cell lymphomas. Anti-CD19 monoclonal antibodies have been generated in mice, and show some promise in pre-clinical animal models of B cell-derived cancers. However, mouse-derived antibodies are generally immunogenic in humans. A number of strategies have been developed to alter mouse-derived antibodies to minimize their immunogenicity in humans. One such strategy, chimerization, involves the fusion of mouse variable regions to human constant regions. However, the mouse-derived variable region sequences remaining following chimerization will often be immunogenic. Another such strategy, humanization, involves the replacement of mouse-derived framework regions (FRs) within the variable regions with the most closely related human-derived sequences, with the optional reversion of certain amino acids back to the corresponding mouse amino acid in order to maintain binding activity. However, even humanized antibodies may be immunogenic, since the antibody complementarity determining regions (CDRs) generally contain B cell epitopes and T cell epitopes that are nonself. Indeed, even fully human antibodies are immunogenic; this is the basis for the formation of anti-idiotype antibodies during the course of an immune response. All of these problems may apply to mouse-derived anti-CD19 antibodies as they would to any other type of antibody. Therefore, there is a need for anti-CD19 antibodies with reduced immunogenicity. Patent Application

[0009] CLIMB -009 / 01 WO 40193 / 34

[0010] IV. Summary of the Invention

[0011] Although the anti-CD19 antibodies with reduced immunogenicity are required for the treatment of patients with those associated conditions, the conventional methods for production of antibodies have some significant shortcomings, including the scalability of such processes.

[0012] The invention provides methods of producing anti-CD19 antibodies. The invention recognizes that the anti-CD19 antibodies will have to be produced at commercially viable scale, especially if an anti -CD 19 antibody is of therapeutic interest for the patients. Accordingly, the present invention is directed to a method or producing an anti-CD19 antibody. In certain aspects, the present invention provides methods and processes for manufacturing the anti-CD19 antibodies in commercially viable scales.

[0013] The invention beneficially recognizes that ADCC activity of the antibody is also influenced by the particular cell line used to produce the antibody. For example, antibodies produced in the mouse myeloma NS / 0 cells (or SP2 / 0 cells) generally have low ADCC, and antibodies produced in rat myeloma Y0 cells (or YB2 / 0) cells have high ADCC (Lifely et al., (1995) Glycobiology 5:813-822).

[0014] In certain embodiments, the invention beneficially allows for the control of Fc-fucosylation levels of the antibody. Attenuation of fucosylation on monoclonal antibodies enhances their potency and has been applied to multiple marketed immunotherapy products. While several bioproduction platforms exist for generating afucosylated antibodies, it remains challenging to precisely control the fucosylation level for optimal pharmacological outcome. Accordingly, in certain embodiments, the invention provides a manufacturing process capable of targeting any predefined fucosylation content in the final product. In certain preferred embodiments, the final product is a fucosylated anti-CD19 antibody.

[0015] In certain embodiments, the platform is based on the CHO DG44 cell line in which the endogenous fucose biosynthesis pathway is genetically abolished. In certain embodiments, the CHO DG44 cell line is GlymaxX® cell line. In certain preferred embodiments, fucosylation content can be fine-tuned by supplementing fucose to the cell growth medium. In certain embodiments, fucose is added in submillimolar concentration of fucose. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0016] In certain preferred embodiments, this system for fucosylation is adopted for clinical-grade production of Budoprutug, an anti-CD19 antibody being developed for B cell immunotherapy. Sine fucosylation is an important aspect of the efficacy of the antibody, it is an important aspect of the production process to closely control and monitor the fucosylation levels of the antibody. In certain embodiments, the fucosylation levels of the anti-CD19 antibody produced by the new CHO DG44 cells is similar to the fucosylation level of the anti-CD19 antibody produced by other cell lines. Advantageously, the process of the invention allows for the control of fucosylation levels by modifying the levels of fucose in the growth medium. In certain exemplary embodiments, fucosylation levels of budoprutug produced by the methods of the current invention are similar to the fucosylation levels of budoprutug produced in YB2 / 0 cells.

[0017] In certain aspects, the present invention provides a composition for preparation of anti- CD19 antibody. In certain embodiments, the invention provides a composition for preparation of an anti -CD 19 antibody, wherein the anti -CD 19 antibody comprises variable domain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and the anti -CD 19 antibody is fucosylated; and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose.

[0018] In certain embodiments, the invention provides a composition for preparation of an anti- CD19 antibody, wherein the anti-CD19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53; and the anti-CD19 antibody is fucosylated; and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose.

[0019] In certain embodiments, the invention provides a composition for preparation of an anti- CD19 antibody, wherein the anti-CD19 antibody comprises the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or more residues corresponding to IlelO, Metl l, Vai 19, Ser51, and Leu53, and Leu53; and the anti-CD19 antibody is fucosylated and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0020] In certain embodiments, the CHO cells are CHO DG44 cells. In certain embodiments, the process does not comprise the use of live viruses and / or infectious retroviral particles. In certain embodiments,

[0021] In certain embodiments, the culture media comprises from about 0.1 mM to about 2.5 mM fucose. In certain embodiments, the culture media comprises from about 0.2 mM to about 2 mM fucose. In certain embodiments, the culture media comprises from about 0.2 mM to about 2 mM fucose. In certain embodiments, the culture media comprises from about 0.25 mM to about 1 mM fucose. In certain embodiments, the culture media comprises about 0.6 mM fucose. In certain embodiments, the culture media comprises about 0.5 mM fucose. In certain embodiments, the culture media comprises about 0.4 mM fucose. In certain embodiments, the culture media comprises about 0.45 mM fucose. In certain embodiments, the culture media comprises about 0.55 mM fucose.

[0022] In certain aspects, the invention provides a process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

[0023] In certain aspects, the invention provides a process for preparation of anti -CD 19 antibody, wherein the anti-CD19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

[0024] In certain aspects, the invention provides a process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0025] In certain embodiments, the anti-CD19 antibody variable domain comprises a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 29. In certain embodiments, the anti-CD19 antibody is provided in U.S. Patent No. 8,691,952, which is incorporated by reference in its entirety. In certain embodiments, the anti-CD19 antibody variable domain comprises a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 29 are provided in U.S. Patent No. 8,691,952.

[0026] In certain embodiments, the heavy chain variable region of the anti-CD19 antibody has, compared to SEQ ID NO: 13, one or more amino acid substitutions selected from the group consisting of Gln5Glu, Ar l9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

[0027] In certain embodiments, the heavy chain variable region of the anti-CD19 antibody is SEQ ID NO: 13 comprising an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93.

[0028] In certain embodiments, the heavy chain variable region of the anti-CD19 antibody has one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

[0029] In certain embodiments, wherein the heavy chain variable region of the anti -CD 19 antibody has one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

[0030] In certain embodiments, wherein the heavy chain variable region of the anti -CD 19 antibody is the amino acid sequence of SEQ ID NO: 17.

[0031] In certain embodiments, the anti -CD 19 antibody variable domain of the anti-CD19 antibody further comprises a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vai 19, Ser51, and Leu53.

[0032] In certain embodiments, wherein the light chain variable region the anti-CD19 antibody has one or more amino acid substitutions selected from the group consisting of IlelOThr, MetllLeu, Vall9Ala, Ser51Asp, and Leu53Thr. Patent Application

[0033] CLIMB -009 / 01 WO 40193 / 34

[0034] In certain embodiments, the light chain variable region is at least 95% identical to SEQ ID NO: 25.

[0035] In certain embodiments, the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, Metl lLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

[0036] In certain embodiments, the light chain variable region is SEQ ID NO: 25 comprising an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53.

[0037] In certain embodiments, the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, Metl lLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

[0038] In certain embodiments, the light chain variable region is the amino acid sequence of SEQ ID NO: 29.

[0039] In certain embodiments, the heavy chain variable region comprises one or more of substitutions Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

[0040] In certain embodiments, wherein the light chain variable region comprises one or more of substitutions IlelOThr, MetllLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

[0041] In certain embodiments, the anti -CD 19 antibody is VB119. In certain embodiments, VB 119 is an anti-CD19, IgGl monoclonal antibody. VB119 demonstrates antibody-dependent cell mediated toxicity (ADCC) with minimal to no complement dependent cytotoxicity (CDC).

[0042] In certain embodiments, the anti-CD19 antibody comprises the below listed subunits in the anti-CD19 antibody:

[0043] Subunit 1 (SEQ ID NO: 50):

[0044] QVQLEQPGAEVVKPGASVKVSCKTSGYTFTSNWMHWVKQTPGKGLEWIGEIDP SDSYTNYNQKFDGKAKLTVDKSSSTAYMEVSDLTAEDSATYYCARGSNPYYYAMDYW GQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV Patent Application CLIMB -009 / 01 WO 40193 / 34

[0045] HTFPAVLQ S SGLYSLS S VVT VP S S SLGTQTYICNVNHKP SNTK VDKRVEPK SCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGK

[0046] Subunit 2 (SEQ ID NO: 51):

[0047] QVQLEQPGAEVVKPGASVKVSCKTSGYTFTSNWMHWVKQTPGKGLEWIGEIDP SDSYTNYNQKFDGKAKLTVDKSSSTAYMEVSDLTAEDSATYYCARGSNPYYYAMDYW GQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP1EKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0048] Subunit 3 (SEQ ID NO: 52):

[0049] QIVLTQSPATLSASPGEKATMTCSASSGVNYMHWYQQKPGTSPKRWIYDTDKTAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRGSYTFGGGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0050] Subunit 4 (SEQ ID NO: 53):

[0051] QIVLTQSPATLSASPGEKATMTCSASSGVNYMHWYQQKPGTSPKRWIYDTDKTAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRGSYTFGGGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0052] In certain embodiments, the heavy chain sequence of the anti-CD19 antibody is provided below (SEQ ID NO: 54):

[0053] QVQLEQPGAEVVKPGASVKVSCKTSGYTFTSNWMHWVKQTPGKGLEWIGEIDP SDSYTNYNQKFDGKAKLTVDKSSSTAYMEVSDLTAEDSATYYCARGSNPYYYAMDYW Patent Application CLIMB -009 / 01 WO 40193 / 34

[0054] GQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0055] In certain embodiments, the light chain sequence of the anti -CD 19 antibody is provided below (SEQ ID NO: 55):

[0056] QIVLTQSPATLSASPGEKATMTCSASSGVNYMHWYQQKPGTSPKRWIYDTDKTAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRGSYTFGGGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0057] In certain embodiments, the anti-CD19 antibody is budoprutug. In certain embodiments, budoprutug comprises variable domain comprises a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 29.

[0058] In certain embodiments, the anti-CD19 antibody is budoprutug. In certain embodiments, budoprutug comprises a heavy chain and a light chain. In certain embodiments, budoprutug comprises heavy chain and light chain provided in SEQ ID NO: 54 and SEQ ID NO: 55 respectively.

[0059] In certain embodiments, the heavy chain of anti-CD19 antibody is at least 90% identical to SEQ ID NO: 54. In certain embodiments, the heavy chain of anti-CD19 antibody is at least 95% identical to SEQ ID NO: 54. In certain embodiments, the heavy chain of anti-CD19 antibody is at least 98% identical to SEQ ID NO: 54. In certain embodiments, the heavy chain of anti-CD19 antibody is identical to SEQ ID NO: 54.

[0060] In certain embodiments, the light chain of anti -CD 19 antibody is at least 90% identical to SEQ ID NO: 55. In certain embodiments, the light chain of anti-CD19 antibody is at least 95% identical to SEQ ID NO: 55. In certain embodiments, the light chain of anti-CD19 antibody is at least 98% identical to SEQ ID NO: 55. In certain embodiments, the light chain of anti-CD19 antibody is identical to SEQ ID NO: 55. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0061] In certain embodiments, the CHO cells in methods of the invention are CHO DG44 cells. In certain embodiments, the CHO DG44 cells advantageously provide optimal production of the anti-CD19 antibodies. In certain beneficial aspects, the methods of the invention do not involve the use of live viruses and / or infectious retroviral particles.

[0062] In certain embodiments, the methods of the invention comprise one or more of the below steps: thawing the CHO cells; culturing the thawed CHO cells; isolate the anti-CD19 antibody; and purify the anti-CD19 antibody.

[0063] Advantageously, the processes of the invention, wherein the anti -CD 19 antibody is produced by CHO cells is advantageous as compared to the processes wherein said antibody is produced by YB2 / 0 cells. In particular, the invention recognized that the processes involving the production of anti-CD19 antibodies in YB2 / 0 cells, would have been unable to prepare the quantities in commercially viable scales.

[0064] In contrast, advantageously, the methods of the invention allow for generation of higher protein expression per cell of anti-CD19 antibodies. In certain preferred embodiments, the methods of the invention lead to production of greater than 3.0 g / L titer of anti-CD19 antibodies. In certain preferred embodiments, the invention beneficially allows for the production of 250L batches of anti-CD19 antibodies. In certain embodiments, the methods of the invention allows for the production of batches of 250L or greater with a titer of 3.0 g / L or higher titer of anti-CD19 antibodies.

[0065] In another beneficial aspect of the invention, the invention provides that even though the anti-CD19 antibody is produced using a new cost efficient and scalable for commercial production, the anti-CD19 antibodies has similar biological activity as compared to the anti-CD19 antibodies produced by other methods. In certain preferred aspects, the biological activity of the anti -CD 19 antibodies produced by methods of the invention has biological activity of at least -75% as compared to the anti-CD19 antibodies produced by other methods.

[0066] The invention beneficially recognizes that ADCC activity of the antibody is also influenced by the particular cell line used to produce the antibody. For example, antibodies produced in the Patent Application CLIMB -009 / 01 WO 40193 / 34 mouse myeloma NS / 0 cells (or SP2 / 0 cells) generally have low ADCC, and antibodies produced in rat myeloma YO cells (or YB2 / 0) cells have high ADCC (Lifely et al., (1995) Glycobiology 5:813-822).

[0067] In certain embodiments, the invention beneficially allows for the control of Fc-fucosylation levels of the antibody.

[0068] In the processes involving the production of anti -CD 19 antibodies in YB2 / 0 cells leads to reduce fucosylation. In particular, the YB2 / 0 cells have reduced fucosylation in the Fc region because the reduced expression of FUT8. The level of fucosylation is an important parameter for the anti -CD 19 antibodies and it impacts the biological activity of the anti-CD19 antibodies.

[0069] In comparison, the methods of the invention advantageously provide an optimal fucosylation pattern of the product. In certain beneficial aspects, the optimal fucosylation product of anti-CD19 antibodies, including VB119 have optimal biological activity.

[0070] In certain aspects, the CHO cells used in the invention express a heterologous enzyme that depletes fucose in the cells. Such cells produce afucosylated antibodies. The level of fucosylation may be modulated by fucose supplementation in the medium in which the cells are cultured. In certain embodiments, the heterologous enzyme expressed in the cell is GDP-6-deoxy-D-lyxo-4- hexulose reductase (RMD). RMD is a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose, and hence results in production of afucosylated antibodies.

[0071] Beneficially, RMD redirects the de-novo fucose synthesis pathway towards a sugarnucleotide (GDP -Rhamnose) that cannot be metabolized by the cell and moreover acts as an inhibitor of the pathway. Even lowest levels expression of this enzyme is sufficient to block the de novo fucose synthetic pathway almost completely. In certain embodiments, the technology used by methods of the invention is provided included in U.S. Patent Nos. 7,579,170, 7,931,895, 7,541,029, 8,124,078, 8,153,124, 8,178,093, 8,409,572, 8,642,292, and 8,357,370 which are incorporated by reference in its entirety.

[0072] In certain embodiments, the culture media comprises from about 0.1 mM to about 2.5 mM fucose. In certain embodiments, the culture media comprises from about 0.2 mM to about 2 mM fucose. In certain embodiments, the culture media comprises from about 0.2 mM to about 2 mM fucose. In certain embodiments, the culture media comprises from about 0.25 mM to about 1 mM Patent Application CLIMB -009 / 01 WO 40193 / 34 fucose. In certain embodiments, the culture media comprises about 0.6 mM fucose. In certain embodiments, the culture media comprises about 0.5 mM fucose. In certain embodiments, the culture media comprises about 0.4 mM fucose. In certain embodiments, the culture media comprises about 0.45 mM fucose. In certain embodiments, the culture media comprises about 0.55 mM fucose.

[0073] In certain embodiments, the CHO cells for manufacturing anti-CD19 antibodies are cultured for from about 5 days to about 30 days. In certain embodiments, the CHO cells for manufacturing anti-CD19 antibodies are cultured for from about 12 days to about 20 days. In certain embodiments, the CHO cells for manufacturing anti-CD19 antibodies are cultured for from about 12 days to about 15 days. In certain embodiments, the CHO cells for manufacturing anti- CD19 antibodies are cultured for about 12 days. In certain embodiments, the CHO cells for manufacturing anti -CD 19 antibodies are cultured for about 13 days. In certain embodiments, the CHO cells for manufacturing anti-CD19 antibodies are cultured for about 14 days. In certain embodiments, the CHO cells for manufacturing anti-CD19 antibodies are cultured for about 15 days.

[0074] The invention beneficially recognizes that attenuation of fucosylation on antibodies may enhance their potency. While several bioproduction platforms exist for generating afucosylated antibodies, it remains challenging to precisely control the fucosylation level for optimal pharmacological outcome. The methods of the invention provide a platform capable of targeting any predefined fucosylation content in the final product. The methods of the invention rely on producing antibodies in cell lines in which the endogenous fucose biosynthesis pathway is genetically abolished. In certain embodiments, the cell line is CHO DG44 GlymaxX® cell line.

[0075] Advantageously, in the methods of the invention, fucosylation content can be fine-tuned by supplementing submillimolar concentration of fucose to the cell growth medium. In certain embodiments, this system was adopted for the clinical production of the anti-CD19 antibodies of the invention which are being developed for B cell immunotherapy.

[0076] As noted herein, the previous methods for production of anti -CD 19 antibodies of the invention relies on CHO cells instead of YB2 / 0 cells to increase the yield of the antibody being produced. However, there was an unmet need to ensure that the anti-CD19 antibodies produced by Patent Application CLIMB -009 / 01 WO 40193 / 34 the new methods have optimal fucosylation to provide acceptable activity and and / or an acceptable therapeutic profile of the antibody.

[0077] The methods of the invention rely on a Design of Experiment (DoE) approach. The DoE approach was implemented in small scale bioreactors to evaluate the combinatorial effects of fucose concentration, medium feed and other bioreactor parameters on product yield and quality. The study established process parameters that lead to >10 X increase in titer and an optimal fucosylation level. Importantly, the processes of the invention are reproducible at large scale. The process is further verified at 250 L and 1000 L scale, demonstrating highly comparable product attributes, such as glycan, charge profile and ADCC potency. Collectively, the methods of the invention provide a scalable solution for producing biotherapeutics with tunable glycan profile, that would unleash the full therapeutic potential of immunotherapies.

[0078] In certain embodiments, the anti-CD19 antibody is about 30% to about 75% fucosylated. In certain embodiments, the anti-CD19 antibody is about 35% to about 70% fucosylated. In certain embodiments, the anti-CD19 antibody is about 40% fucosylated. In certain embodiments, the anti- CD19 antibody is about 45% fucosylated. In certain embodiments, the anti-CD19 antibody is about 50% fucosylated. In certain embodiments, the anti-CD19 antibody is about 55% fucosylated. In certain embodiments, the anti-CD19 antibody is about 60% fucosylated. In certain embodiments, the anti-CD19 antibody is about 65% fucosylated.

[0079] In certain aspects, the invention provides that the anti-CD19 antibody has minimal fucosylation. In these embodiments, the invention provides that the anti-CD19 antibodies have less than 5% fucosylation. In certain embodiments, the invention provides that the anti-CD19 antibodies have less than 4% fucosylation. In certain embodiments, the invention provides that the anti-CD19 antibodies have less than 3% fucosylation. In certain embodiments, the invention provides that the anti -CD 19 antibodies have less than 2% fucosylation. In certain embodiments, the invention provides that the anti-CD19 antibodies have less than 1% fucosylation.

[0080] In certain embodiments, the invention provides that the anti-CD19 antibodies with minimal fucosylation are produced by culturing CHO cells in a media without additional fucose being added to the media. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0081] In another beneficial aspect of the invention, the invention provides that even though the anti-CD19 antibody is produced using a new cost efficient and scalable for commercial production, the anti-CD19 antibodies has similar biological activity as compared to the anti-CD19 antibodies produced by other methods. In certain preferred aspects, the biological activity of the anti -CD 19 antibodies produced by methods of the invention has biological activity of at least -95% as compared to the anti -CD 19 antibodies produced by other methods.

[0082] In certain aspects, the invention provides methods for treatment of a disease by administration of an anti-CD19 antibody. In certain preferred aspects, the anti-CD19 antibody comprises variable domain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93; and the anti-CD19 antibody is prepared by a process comprising production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells. In certain aspects, the methods of the invention provide that the disease to be treated is an autoimmune disorder. In certain embodiments, the autoimmune disorder is primary membranous nephropathy (PMN).

[0083] In certain aspects, the invention provides:

[0084] A process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

[0085] A process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metl l, Vall9, Ser51, and Leu53; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

[0086] A process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or more residues corresponding to IlelO, Metl l, Vall9, Ser51, Patent Application CLIMB -009 / 01 WO 40193 / 34 and Leu53; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

[0087] In certain embodiments, the anti-CD19 antibody comprises a heavy chain variable region comprising SEQ ID NO: 17 and / or a light chain variable region of SEQ ID NO: 29.

[0088] In certain embodiments, wherein the CHO cells are CHO DG44 cells. In certain embodiments, the process comprises one or more of the below steps: thawing the CHO cells; culturing the thawed CHO cells; harvest the cultured CHO cells; isolate the anti-CD19 antibody; and purify the anti-CD19 antibody.

[0089] In certain embodiments, the heavy chain variable region of the anti-CD19 antibody comprises amino acid sequence of SEQ ID NO: 17.

[0090] In certain embodiments, light chain variable region of the anti-CD19 antibody comprises amino acid sequence of SEQ ID NO: 29.

[0091] In certain embodiments, the heavy chain variable region of the anti-CD19 antibody comprises SEQ ID NO: 17 and the light chain variable region comprises SEQ ID NO: 29.

[0092] In certain aspects, the invention provides a method for treatment of a disease by administration of an anti-CD19 antibody, wherein the anti-CD19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53; and the anti -CD 19 antibody is prepared by a process comprising production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

[0093] In certain aspects, the invention provides a method for treatment of a disease by administration of an anti-CD19 antibody, wherein the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53; and the anti-CD19 antibody is prepared by a process comprising production of the anti -CD 19 antibody in Chinese Hamster Ovary (CHO) cells.

[0094] In certain aspects, the invention provides a process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, Patent Application CLIMB -009 / 01 WO 40193 / 34 and Val93; and the process comprises production of the anti-CD19 in cells, and fucosylation of the anti -CD 19 antibody.

[0095] In certain embodiments, the invention provides a process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metl l, Vai 19, Ser51, and Leu53; and the process comprises production of the anti-CD19 in cells, and fucosylation of the anti-CD19 antibody.

[0096] In certain embodiments, the invention provides a process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vai 19, Ser51, and Leu53; and the process comprises production of the anti-CD19 in cells, and fucosylation of the anti-CD19 antibody.

[0097] In certain embodiments, the anti-CD19 antibody variable domain comprises a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 29.

[0098] In certain embodiments, the cells are Chinese Hamster Ovary (CHO) cells. In certain embodiments, the CHO cells are CHO DG44 cells. In certain embodiments, the CHO cells are grown in a media comprising fucose. The quantity of fucose in the media is modulated to achieve optimal level of fucosylation on the anti -CD 19 antibody. In certain embodiments, the media comprises from about 0.2 mM to about 2 mM fucose. In certain embodiments, the media comprises from about 0.1 mM to about 2.5 mM fucose. In certain embodiments, the media comprises from about 0.2 mM to about 1.5 mM fucose. In certain embodiments, the media comprises from about 0.25 mM to about 1 mM fucose. In certain embodiments, the media comprises about 0.5 mM fucose. In certain embodiments, the media comprises about 0.4 mM fucose. In certain embodiments, the media comprises about 0.6 mM fucose. In certain embodiments, the media does not include fucose. In certain embodiments, the cells are cultured from about 5 days to about 30 days. In certain embodiments, the cells are cultured from about 10 days to about 25 days. In certain embodiments, the cells are cultured from about 12 days to about 20 days. In certain embodiments, the cells are cultured from about 12 days to about 15 days. In certain embodiments, the anti-CD19 antibody is 30% to about 75% fucosylated. In certain embodiments, the anti-CD19 antibody is 35% to about 70% fucosylated. In certain embodiments, the anti-CD19 antibody is about 40% Patent Application CLIMB -009 / 01 WO 40193 / 34 fucosylated. In certain embodiments, the anti-CD19 antibody is about 45% or 46% fucosylated. In certain embodiments, the anti-CD19 antibody is about 50% fucosylated. In certain embodiments, the anti-CD19 antibody is about 55% fucosylated. In certain embodiments, the anti -CD 19 antibody is about 60% fucosylated. In certain embodiments, the anti-CD19 antibody is about 65% fucosylated. In certain embodiments, the anti -CD 19 antibody has less than 2% fucosylation.

[0099] In certain embodiments, the volume of media is at least 200L. In certain embodiments, the volume of media is about 250L. In certain embodiments, the volume of media is at least 500L. In certain embodiments, the volume of media is about 500L. In certain embodiments, the volume of media is at least lOOOL. In certain embodiments, the volume of media is about WOOL. In certain embodiments, the volume of media is 200L, 500L, or WOOL.

[0100] In certain aspects, the invention provides a composition for preparation of an anti -CD 19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and the anti-CD 19 antibody is fucosylated; and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose.

[0101] In certain aspects, the invention provides a composition for preparation of an anti -CD 19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IleW, Metll, Vai 19, Ser51, and Leu53; and the antiCD 19 antibody is fucosylated; and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose.

[0102] In certain aspects, the invention provides a composition for preparation of an anti -CD 19 antibody, wherein: the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or more residues corresponding to IleW, Metll, Vall9, Ser51, and Leu53, and Leu53; and the anti-CD19 antibody is fucosylated; and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0103] In certain embodiments, the CHO cells are CHO DG44 cells. In certain embodiments, the CHO cells are CHO DG44 cells. In certain embodiments, the CHO cells are grown in a media comprising fucose. The quantity of fucose in the media is modulated to achieve optimal level of fucosylation on the anti-CD19 antibody. In certain embodiments, the media comprises from about 0.2 mM to about 2 mM fucose. In certain embodiments, the media comprises from about 0.1 mM to about 2.5 mM fucose. In certain embodiments, the media comprises from about 0.2 mM to about 1.5 mM fucose. In certain embodiments, the media comprises from about 0.25 mM to about 1 mM fucose. In certain embodiments, the media comprises about 0.5 mM fucose. In certain embodiments, the media comprises about 0.4 mM fucose. In certain embodiments, the media comprises about 0.6 mM fucose.

[0104] In certain embodiments, the quantity of fucose in the media is a pre-determined amount to achieve optimal level of fucosylation on the anti -CD 19 antibody. In certain embodiments, the predetermined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is 30% to about 75% fucosylated. In certain embodiments, the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is 35% to about 70% fucosylated. In certain embodiments, the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is about 40% fucosylated. In certain embodiments, the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is about 45% or 46% fucosylated. In certain embodiments, the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is about 50% fucosylated. In certain embodiments, the predetermined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is 55% fucosylated. In certain embodiments, the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is 60% fucosylated. In certain embodiments, the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is 65% fucosylated.

[0105] In certain embodiments, the volume of the composition is at least 200L. In certain embodiments, volume of media is about 250L. In certain embodiments, volume of media is at least 500L. In certain embodiments, volume of media is about 500L. In certain embodiments, volume of media is at least 1000L. In certain embodiments, volume of media is about lOOOL. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0106] V. Brief Description of the Drawings

[0107] FIG. 1 provides ADCC data comparison for budoprutug and low-fucose budoprutug as compared to wt-IgG.

[0108] FIG. 2 provides an overview of the control of fucosylation pathway.

[0109] FIG. 3 provides data pertaining to the parameters for scaling up from IL to 250 L scale.

[0110] FIG. 4 provides the data pertaining to cell density, viability, and glycan profiles for the 250L and OOL production.

[0111] FIG. 5 provides process development parameters for the several parameters for IL scale.

[0112] FIG. 6 provides the comparison of properties of budoprutug produced by different processes.

[0113] VI. Detailed Description

[0114] The present invention is directed to processes for preparation of an anti-CD19 murine monoclonal B4 antibody which has been modified to reduce its immunogenicity in comparison to wild-type B4 antibody. More specifically, the variable region of the B4 antibody of the invention is modified to remove potential T-cell epitopes. As a result, B4 antibodies produced by the methods of the invention have improved biological properties compared to wild-type B4 antibodies. More specifically, the mutations within a B4 antibody that have the effect of reducing the immunogenicity of a B4 antibody itself, primarily by removing T-cell epitopes within B4 that may stimulate to an immune response.

[0115] Anti-CD19 antibodies of the invention:

[0116] In certain aspects, the invention provides methods of preparing anti -CD 19 antibodies which feature an amino acid sequence defining a modified immunoglobulin heavy chain framework region comprising amino acid residues 1-30 of SEQ ID NO:22, wherein one or more of the amino acid residues at positions X5, X12, X19, X20, X23, and X24 are as follows: X5 is Q or E, X12 is V or K, X19 is R or K, X20 is L or V, X23 is K, E or D, or X24 is T or A. In certain embodiments, at least one of the amino acid residues at positions X5, X12, X19, X20, X23, or X24 is not the same amino acid residue as the amino acid at the corresponding position in the Patent Application CLIMB -009 / 01 WO 40193 / 34 unmodified immunoglobulin heavy chain framework region as set forth in amino acid residues 1 - 30 of SEQ ID NO: 13. In one embodiment, X23 is E or D.

[0117] In certain aspects, the invention provides methods of preparing anti -CD 19 antibodies which feature an amino acid sequence defining a modified immunoglobulin heavy chain framework region comprising amino acid residues 1-14 of SEQ ID NO:23, wherein one or more of the amino acid residues at positions X3, X5, X7, and X8, are as follows: X3 is K or R, X5 is R, T, or A, X7 is G, D, or E, or X8 is Q or K. According to this aspect of the invention, at least one of the amino acid residues at positions X3, X5, X7, or X8 is not the same as the amino acid at the corresponding position in the unmodified immunoglobulin heavy chain framework region as set forth in amino acid residues 36-49 of SEQ ID NO: 13. In one embodiment, X7 is E or D.

[0118] In certain aspects, the invention provides methods of preparing anti -CD 19 antibodies which feature an amino acid sequence defining a modified immunoglobulin heavy chain framework region comprising amino acid residues 1-39 of SEQ ID NO:24, wherein one or more of the amino acid residues at positions X6, XI 0, X26, X29, and X34 are as follows: X6 is K, D, or E, X10 is K, E, or D, X26 is S, D, or E, X29 is S or A, or X34 is V or T. According to this aspect of the invention, at least one of the amino acid residues at positions X6, X10, X26, X29, or X34 is not the same as the amino acid at the corresponding position in the unmodified immunoglobulin heavy chain framework region as set forth in amino acid residues 60-98 of SEQ ID NO: 13. In one embodiment, X10 is E or D.

[0119] In certain aspects, the invention provides methods of preparing anti -CD 19 antibodies which feature an amino acid sequence defining a modified immunoglobulin light chain framework region comprising amino acid residues 1-23 of SEQ ID NO:32, wherein one or more of the amino acid residues at positions XI, X3, X7, X10, XI 1, and X19 are as follows: XI is Q or D, X3 is V or A, X7 is S or E, XI 0 is I or T, XI 1 is M or L, or XI 9 is V or A. According to this aspect of the invention, at least one of the amino acid residues at positions XI, X3, X7, X10, XI 1, or X19 is not the same as the amino acid at the corresponding position in the unmodified immunoglobulin light chain framework region as set forth in amino acid residues 1-23 of SEQ ID NO:25. In one embodiment, X3 is A and X7 is E. In another embodiment, XI is D, X10 is I, and XI 1 is L. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0120] In another aspect, the invention features an amino acid sequence defining a modified immunoglobulin light chain complementarity determining region comprising amino acid residues 24-33 of SEQ ID NO:28.

[0121] In another aspect, the invention features an amino acid sequence defining a modified immunoglobulin light chain framework region comprising amino acid residues 56-87 of SEQ ID NO:28.

[0122] According to another aspect, the invention features an antibody variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31, wherein the antibody variable region specifically binds to CD 19.

[0123] According to another aspect, the invention features a polypeptide at least 90% or at least 95% identical to a B4 antibody heavy chain variable region, the polypeptide comprising an amino acid substitution at one or more residues corresponding to Vall2, Leu20, Lys23, Thr24, Lys38, Gly42, Gln43, Lys65, Lys69, Ser85, Ser88, or Val93. In one embodiment, the polypeptide comprises one or more of substitutions Gln5Glu, Vall2Lys, Argl9Lys, Leu20Val, Lys23Glu, Lys23Asp, Thr24Ala, Lys38Arg, Arg40Thr, Gly42Asp, Gly42Glu, Gln43Lys, Lys65Asp, Lys65Glu, Lys69Glu, Lys69Asp, Ser85Asp, Ser85Glu, Ser88Ala, or Val93Thr.

[0124] According to another aspect, the invention features a polypeptide at least 90% or at least 95% identical to a B4 antibody light chain variable region, the polypeptide comprising an amino acid substitution at one or more residues corresponding to Val3, Ser7, IlelO, Metll, Vai 19, Val29, Ser51, Leu53, Ala54, or Ser75. In one embodiment, the polypeptide comprises one or more of substitutions GlnlAsp, Val3Ala, Ser7Glu, IlelOThr, MetllLeu, Vall9Ala, Val29Ala, Ser51Asp, Leu53Thr, Ala54Asp, or Ser75Glu.

[0125] In another aspect, the invention features a method of treating a patient, the method comprising the step of administering a therapeutically effective amount of a anti-CD19 prepared by methods provided in the invention.

[0126] In another aspect, the invention features a method for targeting a cell with CD 19 on its surface, the method comprising the step of administering an antibody variable region according to Patent Application CLIMB -009 / 01 WO 40193 / 34 any one of the embodiments of the invention. In one embodiment of the method the cell is a tumor cell.

[0127] In certain aspects, the invention provides a process for preparation of anti -CD 19 antibody, wherein the anti-CD19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vai 19, Ser51, and Leu53; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

[0128] In certain aspects, the invention provides a process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vai 19, Ser51, and Leu53; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

[0129] In certain embodiments, the anti-CD19 antibody variable domain comprises a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 29. In certain embodiments, the anti-CD19 antibody is provided in U.S. Patent No. 8,691,952, which is incorporated by reference in its entirety. In certain embodiments, the anti-CD19 antibody variable domain comprises a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 29 are provided in U.S. Patent No. 8,691,952.

[0130] In certain embodiments, the heavy chain variable region of the anti-CD19 antibody has, compared to SEQ ID NO: 13, one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

[0131] In certain embodiments, the heavy chain variable region of the anti-CD19 antibody is SEQ ID NO: 13 comprising an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93.

[0132] In certain embodiments, the heavy chain variable region of the anti -CD 19 antibody has one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0133] In certain embodiments, wherein the heavy chain variable region of the anti-CDl 9 antibody has one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

[0134] In certain embodiments, wherein the heavy chain variable region of the anti -CD 19 antibody is the amino acid sequence of SEQ ID NO: 17.

[0135] In certain embodiments, the anti -CD 19 antibody variable domain of the anti-CDl 9 antibody further comprises a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vai 19, Ser51, and Leu53.

[0136] In certain embodiments, wherein the light chain variable region the anti-CDl 9 antibody has one or more amino acid substitutions selected from the group consisting of IlelOThr, MetllLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

[0137] In certain embodiments, the light chain variable region is at least 95% identical to SEQ ID NO: 25.

[0138] In certain embodiments, the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, Metl lLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

[0139] In certain embodiments, the light chain variable region is SEQ ID NO: 25 comprising an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vai 19, Ser51, and Leu53.

[0140] In certain embodiments, the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, Metl lLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

[0141] In certain embodiments, the light chain variable region is the amino acid sequence of SEQ ID NO: 29.

[0142] In certain embodiments, the heavy chain variable region comprises one or more of substitutions Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0143] In certain embodiments, wherein the light chain variable region comprises one or more of substitutions IlelOThr, MetllLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

[0144] In certain embodiments, the anti -CD 19 antibody is VB119. In certain embodiments, VB 119 is an anti-CD19, IgGl monoclonal antibody. VB119 demonstrates antibody-dependent cell mediated toxicity (ADCC) with minimal to no complement dependent cytotoxicity (CDC).

[0145] In certain embodiments, the anti-CD19 antibody comprises one or more of the below listed subunits in the anti -CD 19 antibody:

[0146] Subunit 1 (SEQ ID NO: 50):

[0147] QVQLEQPGAEVVKPGASVKVSCKTSGYTFTSNWMHWVKQTPGKGLEWIGEIDP SDSYTNYNQKFDGKAKLTVDKSSSTAYMEVSDLTAEDSATYYCARGSNPYYYAMDYW GQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0148] Subunit 2 (SEQ ID NO: 51):

[0149] QVQLEQPGAEVVKPGASVKVSCKTSGYTFTSNWMHWVKQTPGKGLEWIGEIDP SDSYTNYNQKFDGKAKLTVDKSSSTAYMEVSDLTAEDSATYYCARGSNPYYYAMDYW GQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0150] Subunit 3 (SEQ ID NO: 52):

[0151] QIVLTQSPATLSASPGEKATMTCSASSGVNYMHWYQQKPGTSPKRWIYDTDKTAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRGSYTFGGGTKLEIKRTVAAPSVFIFP Patent Application

[0152] CLIMB -009 / 01 WO 40193 / 34

[0153] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

[0154] LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0155] Subunit 4 (SEQ ID NO: 53):

[0156] QIVLTQSPATLSASPGEKATMTCSASSGVNYMHWYQQKPGTSPKRWIYDTDKTAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRGSYTFGGGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0157] In certain embodiments, the heavy chain sequence of the anti-CD19 antibody is provided below (SEQ ID NO: 54):

[0158] QVQLEQPGAEVVKPGASVKVSCKTSGYTFTSNWMHWVKQTPGKGLEWIGEIDP SDSYTNYNQKFDGKAKLTVDKSSSTAYMEVSDLTAEDSATYYCARGSNPYYYAMDYW GQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0159] In certain embodiments, the light chain sequence of the anti -CD 19 antibody is provided below (SEQ ID NO: 55):

[0160] QIVLTQSPATLSASPGEKATMTCSASSGVNYMHWYQQKPGTSPKRWIYDTDKTAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRGSYTFGGGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0161] In certain embodiments, the anti-CD19 antibody is budoprutug. In certain embodiments, budoprutug comprises a heavy chain and a light chain. In certain embodiments, budoprutug comprises heavy chain and light chain provided in SEQ ID NO: 54 and SEQ ID NO: 55 respectively. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0162] In certain embodiments, the heavy chain of anti-CDl 9 antibody is at least 90% identical to SEQ ID NO: 54. In certain embodiments, the heavy chain of anti-CD19 antibody is at least 95% identical to SEQ ID NO: 54. In certain embodiments, the heavy chain of anti-CDl 9 antibody is at least 98% identical to SEQ ID NO: 54. In certain embodiments, the heavy chain of anti-CD19 antibody is identical to SEQ ID NO: 54.

[0163] In certain embodiments, the light chain of anti -CD 19 antibody is at least 90% identical to SEQ ID NO: 55. In certain embodiments, the light chain of anti-CD19 antibody is at least 95% identical to SEQ ID NO: 55. In certain embodiments, the light chain of anti-CD19 antibody is at least 98% identical to SEQ ID NO: 55. In certain embodiments, the light chain of anti-CD19 antibody is identical to SEQ ID NO: 55.

[0164] Fc Portion

[0165] In certain embodiments, the antibody variable domains of the antibodies prepared using the methods of the invention are optionally fused to an Fc portion. As used herein, the Fc portion encompasses domains derived from the heavy chain constant region of an immunoglobulin, preferably a human immunoglobulin, including a fragment, analog, variant, mutant or derivative of the constant region. The constant region of an immunoglobulin heavy chain is defined as a naturally-occurring or synthetically produced polypeptide homologous to at least a portion of the C-terminal region of the heavy chain, including the CHI, hinge, CH2, CH3, and, for some heavy chain classes, CH4 domains. The “hinge” region joins the CHI domain to the CH2-CH3 region of an Fc portion. The constant region of the heavy chains of all mammalian immunoglobulins exhibit extensive amino acid sequence similarity.

[0166] In the present invention, the Fc portion typically includes at least a CH2 domain. For example, the Fc portion can include the entire immunoglobulin heavy chain constant region (CH1- hinge-CH2-CH3). Alternatively, the Fc portion can include all or a portion of the hinge region, the CH2 domain and the CH3 domain.

[0167] The constant region of an immunoglobulin is responsible for many important antibody effector functions, including those mediated by Fc receptor (FcR) binding and by complement binding. There are five major classes of the heavy chain constant region, classified as IgA, IgG, IgD, IgE, and IgM, each with characteristic effector functions designated by isotype. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0168] IgG, for example, is separated into four y isotypes: yl , y2, y3, and y4, also known as IgGl , IgG2, IgG3, and IgG4, respectively. IgG molecules can interact with multiple classes of cellular receptors including three classes of Fey receptors (FcyR) specific for the IgG class of antibody, namely FcyRI, FcyRII, and FcyRIII. The sequences important for the binding of IgG to the FcyR receptors have been reported to be in the CH2 and CH3 domains.

[0169] It is also often useful to alter the serum half-life of the antibody. The serum half-life of an antibody, as of an immunoglobulin fusion protein, is influenced by the ability of that antibody to bind to an Fc receptor (FcR) (Gillies et al., Cancer Research (1999) 59:2159-66). The CH2 and CH3 domains of IgG2 and IgG4 have undetectable or reduced binding affinity to Fc receptors compared to those of IgGl. Accordingly, the serum half-life of the featured antibody can be increased by using the CH2 and / or CH3 domain from IgG2 or IgG4 isotypes. Alternatively, the antibody can include a CH2 and / or CH3 domain from IgGl or IgG3 with modification in one or more amino acids in these domains to reduce the binding affinity for Fc receptors (see, e.g., U.S. patent application Ser. No. 09 / 256,156, published as U.S. patent application publication 2003- 0105294).

[0170] In certain embodiments, an Fc portion fused to an antibody variable region of the invention can contain CH2 and / or CH3 domains and a hinge region that are derived from different antibody isotypes. For example, the Fc portion can contain CH2 and / or CH3 domains of IgG2 or IgG4 and a hinge region of IgGl. Assembly of such hybrid Fc portions has been described in U.S. patent application publication 2003-0044423.

[0171] When fused to an antibody variable region of the invention, the Fc portion may contain one or more amino acid modifications that generally extend the serum half-life of an Fc fusion protein. Such amino acid modifications include mutations substantially decreasing or eliminating Fc receptor binding or complement fixing activity. For example, one type of such mutation removes the glycosylation site of the Fc portion of an immunoglobulin heavy chain. In IgGl, the glycosylation site is Asn297 (see, for example, U.S. patent application Ser. No. 10 / 310,719, published as U.S. patent application publication 2003-0166163).

[0172] Methods of production of the anti-CD19 antibodies: Patent Application CLIMB -009 / 01 WO 40193 / 34

[0173] The contemporary methods of production of antibodies leads to a limited titer of antibodies and a low quantity of the antibody being produced. In certain aspects, the present invention provides methods for preparation of anti -CD 19 antibodies. In certain embodiments, the methods of preparation of anti -CD 19 antibodies result in production of anti-CD19 antibodies that could be manufactured efficiently and maintain the pertinent ADCC profile responsible for the potency of anti-CD19 antibodies.

[0174] The invention recognizes that the anti-CD19 antibodies will have to be produced at commercially viable scale, especially if an anti-CD19 antibody is of therapeutic interest for the patients. Accordingly, the present invention is directed to a method or producing an anti-CD19 antibody monoclonal antibody. In certain aspects, the present invention provides methods and processes for manufacturing the anti -CD 19 antibodies in commercially viable scales.

[0175] The invention provides that the selection of cell lines for biopharmaceutical production is a pivotal decision in the development process, as it profoundly impacts product quality, efficiency, and safety of the anti -CD 19 antibody. The invention beneficially provides that production of the YB2 / 0 cell line has limitations for producing the anti-CD 19 antibodies of the invention. In contrast, production of anti-CD19 antibodies enhances the quality and quantity of anti-CD19 antibodies for the patients.

[0176] In certain aspects, the invention provides a process for preparation of an anti-CD19 antibody, wherein: the anti-CD 19 antibody comprises variable domain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

[0177] Advantageously, the processes of the invention, wherein the anti -CD 19 antibody is produced by CHO cells is advantageous over processes of the invention wherein the antibody is produced by YB2 / 0 cells. In particular, the invention recognized that the processes involving the production of anti-CD 19 antibodies in YB2 / 0 cells, would have been unable to prepare the quantities in commercially viable scales. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0178] In contrast, advantageously, the methods of the invention allow for generation of higher protein expression of anti-CD19 antibodies. In certain preferred embodiments, the methods of the invention lead to production of greater than 3.0 g / L titer of anti-CD19 antibodies. In certain preferred embodiments, the invention beneficially allows for the production of 250L batches of anti-CD19 antibodies. In certain embodiments, the methods of the invention allow for the production of batches of 250L or greater with a titer of 3.0 g / L or higher titer of anti -CD 19 antibodies. In other beneficial aspects of the invention, the invention provides that even though the anti-CD19 antibody is produced using a new cost efficient and scalable for commercial production, the anti-CD19 antibodies has similar biological activity as compared to the anti-CD19 antibodies produced by other methods. In certain preferred aspects, the biological activity of the anti-CD19 antibodies produced by methods of the invention has biological activity of at least -95% as compared to the anti -CD 19 antibodies produced by other methods.

[0179] In certain embodiments, the CHO cells in methods of the invention are CHO DG44 cells. In certain embodiments, the CHO DG44 cells advantageously provide optimal production of the anti-CD19 antibodies. In certain beneficial aspects, the methods of the invention do not involve the use of live viruses and / or infectious retroviral particles.

[0180] In certain embodiments, the methods of the invention comprise one or more of the below steps: thawing the CHO cells; culturing the thawed CHO cells; harvest the cultured CHO cells; isolate the anti-CD19 antibody; and purify the anti-CD19 antibody.

[0181] FIG. 3 provides the data pertaining to the different parameters for the 250L scale. FIG. 4 provides the data pertaining to cell density, viability, and glycan profiles for the 250L and OOL production. The table below provides a side-by-side comparison of the process of preparation of anti-CD19 antibodies in YB2 / 0 and CHO cells are provided below. Patent Application

[0182] CLIMB -009 / 01 WO 40193 / 34 Patent Application CLIMB -009 / 01 WO 40193 / 34

[0183] As highlighted in the above table, the CHO DG44 cells are free of adventitious agents as compared to the process of the YB2 / 0 cells. In certain embodiments, according to the methods of the invention, the medium to culture CHO DG44 cells comprises fucose. The invention recognizes that by adding fucose to the medium, modulation of the mAb fucosylation was achieved.

[0184] Fucosylation

[0185] The invention beneficially recognizes that ADCC activity of the antibody is also influenced by the particular cell line used to produce the antibody. For example, antibodies produced in the mouse myeloma NS / 0 cells (or SP2 / 0 cells) generally have low ADCC, and antibodies produced in rat myeloma YO cells (or YB2 / 0) cells have high ADCC (Lifely et al., (1995) Glycobiology 5:813-822).

[0186] The invention further provides that the type of cell line used for antibody expression affects the carbohydrate structure of the N-linked glycosyl chain, which is attached to the Fc region of the antibody at position corresponding to N297 in IgGl. In certain embodiments, the invention recognizes that the carbohydrate structure of antibodies produced in CHO cells is fucosylated, whereas the carbohydrate chain of antibodies produced in YB2 / 0 is largely absent of fucose. Antibodies that lack fucose on the carbohydrate structure bind to human FcyRIIIa with higher affinity (Shields et al., (2002) JBC 277:26733-26740). In certain embodiments, anti-CD19 antibodies with variable regions of the invention are characterized by having reduced fucosylation on the N-linked glycosyl chain of the Fc portion of the antibody.

[0187] In certain beneficial aspects of the invention, the methods used herein provide that if fucose is absent from the media in which cells are cultured, the CHO cells are unable to synthesize GDP- fucose, and effectively resulting in production of afucosylated antibodies. In effect, the invention provides mechanisms to control the level of fucosylation in the antibodies produced by the methods of the invention.

[0188] FIG. 2 provides a schematic representation of the fucosylation pathway in the cells and the rescue pathway utilized by the methods of the invention. In certain aspects, the CHO cells used in the invention express a heterologous enzyme that depletes fucose in the cells. Such cells produce afucosylated antibodies. The level of fucosylation may be modulated by fucose supplementation in the medium in which the cells are cultured. In certain embodiments, the heterologous enzyme Patent Application CLIMB -009 / 01 WO 40193 / 34 expressed in the cell is GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). RMD is a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose, and hence results in production of afucosylated antibodies. The invention beneficially recognizes that this blocked fucosylation can be rescued by supplementing fucose in the cell culture medium. The invention further recognizes that the fucosylation level on the protein product can be tuned by adjusting the exogenous fucose concentration.

[0189] In certain embodiments, the invention beneficially allows for the control of Fc-fucosylation levels of the antibody.

[0190] In the processes involving the production of anti -CD 19 antibodies in YB2 / 0 cells leads to reduction of fucosylation. In particular, the YB2 / 0 cells have reduced fucosylation in the Fc region because the reduced expression of FUT8 enzyme. The level of fucosylation is an important parameter for the anti-CD19 antibodies and it impacts the biological activity of the anti-CD19 antibodies.

[0191] In comparison, the methods of the invention advantageously provide an optimal fucosylation pattern of the product. In certain beneficial aspects, the optimal fucosylation product of anti-CD19 antibodies, including VB119 have optimal biological activity.

[0192] In certain embodiments, the methods of the invention rely on production of anti-CD19 antibodies using a technology for optimal production and fucosylation of antibodies. In certain embodiments, the technology provides heterologous, stable, cytosolic expression of the bacterial enzyme GDP-4keto-6-deoxy-D-mannose reductase (RMD) in a cell that is intended to be used for production of a glycoprotein or an antibody by subsequent (or simultaneous) transduction of respective genes of the glycoprotein / antibody.

[0193] Beneficially, RMD redirects the de-novo fucose synthesis pathway towards a sugarnucleotide (GDP -Rhamnose) that cannot be metabolized by the cell and moreover acts as an inhibitor of the pathway. Even lowest levels expression of this enzyme is sufficient to block the de novo fucose synthetic pathway almost completely. In certain embodiments, the technology used by methods of the invention is provided included in U.S. Patent Nos. 7,579,170, 7,931,895, 7,541,029, 8,124,078, 8,153,124, 8,178,093, 8,409,572, 8,642,292, and 8,357,370 which are incorporated by reference in its entirety. Patent Application

[0194] CLIMB -009 / 01 WO 40193 / 34

[0195] The invention further provides that the level of fucosylation may be modulated by varying the amount of fucose in the cell medium. As an example, Table 2, included below, the level of fucosylation of the exemplary anti -CD 19 antibodies and their relative potencies.

[0196] Table 2: Fucosylation and Relative Potency

[0197] As provided above, the methods of the invention provide methods of modulating the level of fucosylation on the anti -CD 19 antibody, by adjusting the amount of fucose in the culture medium, and the amount of time that the cells are cultured in such a media.

[0198] The invention beneficially recognizes that attenuation of fucosylation on antibodies may enhance their potency. While several bioproduction platforms exist for generating afucosylated antibodies, it remains challenging to precisely control the fucosylation level for optimal pharmacological outcome. The methods of the invention provide a platform capable of targeting any predefined fucosylation content in the final product. The methods of the invention rely on producing antibodies in cell lines in which the endogenous fucose biosynthesis pathway is genetically abolished. In certain embodiments, the cell line is CHO DG44 GlymaxX® cell line.

[0199] Advantageously, in the methods of the invention, fucosylation content can be fine-tuned by supplementing submillimolar concentration of fucose to the cell growth medium. In certain embodiments, this system was adopted for the clinical production of the anti-CD19 antibodies of the invention which are being developed for B cell immunotherapy. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0200] As noted herein, the previous methods for production of anti-CD19 antibodies of the invention relies on CHO cells instead of YB2 / 0 cells to increase the yield of the antibody being produced. However, there was an unmet need to ensure that the anti-CD19 antibodies produced by the new methods have optimal fucosylation to provide acceptable activity and and / or an acceptable therapeutic profile of the antibody.

[0201] The methods of the invention rely on a Design of Experiment (DoE) approach. The DoE approach was implemented in small scale bioreactors to evaluate the combinatorial effects of fucose concentration, medium feed and other bioreactor parameters on product yield and quality. The study established process parameters that lead to >10 X increase in titer and an optimal fucosylation level. Importantly, the processes of the invention are reproducible at large scale. The process is further verified at 250 L and 1000 L scale, demonstrating highly comparable product attributes, such as glycan, charge profile and ADCC potency. Collectively, the methods of the invention provide a scalable solution for producing biotherapeutics with tunable glycan profile, that would unleash the full therapeutic potential of immunotherapies.

[0202] In certain embodiments, the culture media comprises from about 0.1 mM to about 2.5 mM fucose. In certain embodiments, the culture media comprises from about 0.2 mM to about 2 mM fucose. In certain embodiments, the culture media comprises from about 0.2 mM to about 2 mM fucose. In certain embodiments, the culture media comprises from about 0.25 mM to about 1 mM fucose. In certain embodiments, the culture media comprises about 0.6 mM fucose. In certain embodiments, the culture media comprises about 0.5 mM fucose. In certain embodiments, the culture media comprises about 0.4 mM fucose. In certain embodiments, the culture media comprises about 0.45 mM fucose. In certain embodiments, the culture media comprises about 0.55 mM fucose.

[0203] In certain embodiments, the anti-CD19 antibody of the invention, wherein the anti-CD19 antibody of the invention with low fucose concentration has different activity as compared to budoprutug. An example of the activity of ADCC of the low-fucose budoprutug and budoprutug as compared to wt-IgGl is provided in FIG. 1.

[0204] In certain embodiments, the CHO cells for manufacturing anti-CD19 antibodies are cultured from about 5 days to about 30 days. In certain embodiments, the CHO cells for manufacturing anti-CD19 antibodies are cultured for from about 12 days to about 20 days. In Patent Application CLIMB -009 / 01 WO 40193 / 34 certain embodiments, the CHO cells for manufacturing anti-CDl 9 antibodies are cultured for from about 12 days to about 15 days. In certain embodiments, the CHO cells for manufacturing anti- CD19 antibodies are cultured for about 12 days. In certain embodiments, the CHO cells for manufacturing anti -CD 19 antibodies are cultured for about 13 days. In certain embodiments, the CHO cells for manufacturing anti-CD19 antibodies are cultured for about 14 days. In certain embodiments, the CHO cells for manufacturing anti-CDl 9 antibodies are cultured for about 15 days.

[0205] In certain embodiments, the anti-CD19 antibody is about 30% to about 75% fucosylated. In certain embodiments, the anti-CDl 9 antibody is about 35% to about 70% fucosylated. In certain embodiments, the anti-CDl 9 antibody is about 40% fucosylated. In certain embodiments, the anti- CD19 antibody is about 45% fucosylated. In certain embodiments, the anti-CD19 antibody is about 50% fucosylated. In certain embodiments, the anti-CDl 9 antibody is about 55% fucosylated. In certain embodiments, the anti-CDl 9 antibody is about 60% fucosylated. In certain embodiments, the anti -CD 19 antibody is about 65% fucosylated.

[0206] In certain aspects, the invention provides that the anti-CDl 9 antibody has minimal fucosylation. In these embodiments, the invention provides that the anti-CDl 9 antibodies have less than 5% fucosylation. In certain embodiments, the invention provides that the anti -CD 19 antibodies have less than 4% fucosylation. In certain embodiments, the invention provides that the anti-CDl 9 antibodies have less than 3% fucosylation. In certain embodiments, the invention provides that the anti -CD 19 antibodies have less than 2% fucosylation. In certain embodiments, the invention provides that the anti-CD19 antibodies have less than 1% fucosylation.

[0207] In certain embodiments, the invention provides that the anti-CDl 9 antibodies with minimal fucosylation are produced by culturing CHO cells in a media without additional fucose being added to the media.

[0208] In certain embodiments, the process parameters including pH profile, feeding regime, seeding density, fucose concentration, and dissolved oxygen are optimized for the antibody production. The data provided herein demonstrates systematic evaluation of these parameters for their impact on cell growth and productivity, as well as fucosylation level and potency. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0209] FIG. 5 provides process development parameters for the several parameters for IL scale. The parameters analyzed for developing a process include pH profile, feeding regime, seeding density, fucose concentration, and dissolved oxygen. As the data provided in FIG. 5 demonstrates, the variation of parameters lead to different ADCC levels for the budoprutug antibody produced by the methods of the invention.

[0210] FIG. 6 provides data related to the properties of budoprutug antibody produced from methods of the invention (provided in pink) as compared to the budoprutug produced by the YB2 / 0 cells. Indeed, the processes of the invention lead to increase of titers as compared to the titers produced by the antibody produced in YB2 / 0 cells. In certain embodiments, the methods lead to a two-fold increase in budoprutug titer as compared to the budoprutug produced in YB2 / 0 cells. In certain embodiments, the methods lead to a four-fold increase in budoprutug titer as compared to the budoprutug produced in YB2 / 0 cells. In certain embodiments, the methods lead to a five-fold increase in budoprutug titer as compared to the budoprutug produced in YB2 / 0 cells. In certain embodiments, the methods lead to a ten-fold increase in budoprutug titer as compared to the budoprutug produced in YB2 / 0 cells. In certain embodiments, the methods lead to a twenty-fold increase in budoprutug titer as compared to the budoprutug produced in YB2 / 0 cells.

[0211] In addition, in certain embodiments, the methods of the current invention may be scaled up to at least up to 250L batch size for production of the anti -CD 19 antibody. In addition, in certain embodiments, the methods of the current invention may be scaled up to at least up to 500L of the anti -CD 19 antibody. In addition, in certain embodiments, the methods of the current invention may be scaled up to at least up to 750L batch size for production of the anti-CD19 antibody. In addition, in certain embodiments, the methods of the current invention may be scaled up to at least up to WOOL of the anti-CD19 antibody.

[0212] Methods of treatment:

[0213] In certain aspects, the invention provides methods of treatment of disease by administration of anti -CD 19 antibody, wherein the anti-CD19 antibody is prepared by the methods provided herein.

[0214] In certain preferred embodiments, the methods of the invention provide that the disease to be treated with an anti -CD 19 antibody of the invention is an autoimmune disease. In certain Patent Application CLIMB -009 / 01 WO 40193 / 34 preferred embodiments, the methods of the invention provide that the disease to be treated with an anti-CD19 antibody of the invention is an autoimmune disorder.

[0215] The autoimmune disorder can be a systemic autoimmune disorder or an organ-specific autoimmune disorder. Non-limiting examples of an autoimmune disorder that can be treated using the anti -CD 19 antibodies disclosed herein include an acute disseminated encephalomyelitis (ADEM), an Addison’s disease, an allergy, allergic rhinitis, an Alzheimer’s disease, an antiphospholipid antibody syndrome (APS), an arthritis such as, e.g., a monoarthritis, an oligoarthritis, or a polyarthritis like an osteoarthritis, a rheumatoid arthritis, a juvenile idiopathic arthritis, a septic arthritis, a spondyloarthropathy, a gout, a pseudogout, or Still’s disease, an asthma, an autoimmune deficiency syndrome (AIDS), an autoimmune hemolytic anemia, an autoimmune hepatitis, an autoimmune inner ear disease, a bullous pemphigoid, a celiac disease, a Chagas disease, a chronic obstructive pulmonary disease (COPD), a diabetes mellitus type 1 (IDDM), an endometriosis, a gastrointestinal disorder such as, e.g., an irritable bowel disease or an inflammatory bowel disease like Crohn’s disease or an ulcerative colitis, a Goodpasture’s syndrome, a Graves’ disease, a Guillain-Barre syndrome (GBS), a Hashimoto's thyroiditis, a hidradenitis suppurativa, an idiopathic thrombocytopenic purpura, an interstitial cystitis, a lupus, such as, e.g., a discoid lupus erythematosus, a drug-induced lupus erythematosus, a lupus nephritis, a neonatal lupus, a subacute cutaneous lupus erythematosus, or a systemic lupus erythematosus, a morphea, a multiple sclerosis (MS), a myasthenia gravis, a myopathy such as, e.g., a dermatomyositis, an inclusion body myositis, or a polymyositis, a myositis, a narcolepsy, a neuromyotonia, a Parkinson's disease, a pemphigus vulgaris, a pernicious anemia, a primary biliary cirrhosis, a psoriasis, a recurrent disseminated encephalomyelitis, a rheumatic fever, a schizophrenia, a scleroderma, a Sjogren's syndrome, a skin disorder such as, e.g., dermatitis, an eczema, a statis dermatitis, a hidradenitis suppurativa, a psoriasis, a rosacea or a scleroderma, a tenosynovitis, a uveitis, vasculitis such as, e.g., a Buerger's disease, a cerebral vasculitis, a Churg-Strauss arteritis, a cryoglobulinemia, an essential cryoglobulinemic vasculitis, a giant cell arteritis, a Golfer's vasculitis, a Henoch- Schonlein purpura, a hypersensitivity vasculitis, a Kawasaki disease, a microscopic polyarteritis / polyangiitis, a polyarteritis nodosa, a polymyalgia rheumatica (PMR), a rheumatoid vasculitis, a Takayasu arteritis, or a Wegener's granulomatosis, or a vitiligo. Non-limiting examples of a symptom reduced by a method of treating an autoimmune disorder disclosed herein include inflammation, fatigue, dizziness, malaise, elevated fever and high body temperature, extreme Patent Application

[0216] CLIMB -009 / 01 WO 40193 / 34 sensitivity to cold in the hands and feet, weakness and stiffness in muscles and joints, weight changes, digestive or gastrointestinal problems, low or high blood pressure, irritability, anxiety, or depression, infertility or reduced sex drive (low libido), blood sugar changes, and depending on the type of autoimmune disease, an increase in the size of an organ or tissue, or the destruction of an organ or tissue. Non-limiting examples of an inflammation symptom reduced by a method of treating an autoimmune disorder disclosed herein include edema, hyperemia, erythema, bruising, tenderness, stiffness, swollenness, fever, a chill, congestion of the respiratory tract including nose, and bronchi, congestion of a sinus, a breathing problem, fluid retention, a blood clot, a loss of appetite, an increased heart rate, a formation of granulomas, fibrinous, pus, or non-viscous serous fluid, a formation of an ulcer, or pain.

[0217] In certain embodiments, the autoimmune disease or disorder is primary membranous nephropathy (PMN). Primary membranous nephropathy (PMN) is a kidney-specific, autoimmune glomerular disease that presents with increased protein in the urine associated with a pathognomonic pattern of injury in glomeruli. PMN is the commonest cause of idiopathic nephrotic syndrome in nondiabetic adults worldwide, representing between 20% and 37% in most series and rising to as high as 40% in adults over 60. In certain embodiments, the invention provides that the anti-CD19 antibodies prepared using the methods of the invention are administered for the treatment of PMN.

[0218] In certain embodiments, the methods of the invention provide that the disease to be treated with an anti-CD19 antibody of the invention is cancer. In certain embodiments, the cancer could be any cancer caused by B cells, such as B cell lymphoma.

[0219] Methods of administration:

[0220] The antibodies of the invention are preferably used to treat patients with B cell disorders such as autoimmune disorders with a B cell component such as rheumatoid arthritis, myasthenia gravis, multiple sclerosis, systemic lupus erythematosus.

[0221] In the case of antibodies directed against CD 19, it is sometimes useful to clear the normal B cells from the body, as these cells are likely to titrate the antibody of the invention. Rituxan™ may be used for this purpose, according to standard procedures. Alternatively, the anti-CD 19 antibodies of the invention may be used to clear the normal B cells from the body. Patent Application CLIMB -009 / 01 WO 40193 / 34

[0222] In certain embodiments, the invention provides that the antibodies prepared by the methods of the invention may be administered parenterally. In certain embodiments, the invention provides that the anti-CD19 antibodies of the invention may be administered as an intravenous infusion. Other methods of administration include injection routes such as subcutaneous, intradermal, intramuscular, intraperitoneal, or intravenous (bolus) delivery. Inhalation and oral delivery are also possible methods of delivery.

[0223] For a 70 kilogram human, a typical dose is in the range of about 50 milligrams to 2 grams, with a preferred dose in the range of about 400-600 milligrams. Dosing may be repeated about once every three to six weeks.

[0224] Pharmaceutical compositions of the invention may be used in the form of solid, semi solid, or liquid dosage forms, such as, for example, pills, capsules, powders, liquids, suspensions, or the like, preferably in unit dosage forms suitable for administration of precise dosages. The compositions include a conventional pharmaceutical carrier or excipient and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc. Such excipients may include other proteins, such as, for example, human serum albumin or plasma proteins. Actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art. The composition or formulation to be administered will, in any event, contain a quantity of the active component(s) in an amount effective to achieve the desired effect in the subject being treated.

[0225] Administration of the compositions hereof can be via any of the accepted modes of administration for agents that exhibit such activity. These methods local or systemic administration. Intravenous injection in a pharmaceutically acceptable carrier is a preferred method of administration. The amount of active compound administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.

[0226] Compositions for production:

[0227] In certain aspects, the present invention provides a composition for preparation of anti- CD19 antibody. In certain embodiments, the invention provides a composition for preparation of an anti-CD19 antibody, wherein the anti-CD19 antibody comprises variable domain comprising a Patent Application CLIMB -009 / 01 WO 40193 / 34 heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and the anti -CD 19 antibody is fucosylated; and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose.

[0228] In certain embodiments, the invention provides a composition for preparation of an anti- CD19 antibody, wherein the anti -CD 19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53; and the anti-CD19 antibody is fucosylated; and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose.

[0229] In certain embodiments, the invention provides a composition for preparation of an anti- CD19 antibody, wherein the anti-CD19 antibody comprises the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or more residues corresponding to IlelO, Metl l, Vai 19, Ser51, and Leu53, and Leu53; and the anti-CD19 antibody is fucosylated and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose.

[0230] In certain embodiments, the CHO cells are CHO DG44 cells. In certain embodiments, the process does not comprise the use of live viruses and / or infectious retroviral particles. In certain embodiments,

[0231] In certain embodiments, the culture media comprises from about 0.1 mM to about 2.5 mM fucose. In certain embodiments, the culture media comprises from about 0.2 mM to about 2 mM fucose. In certain embodiments, the culture media comprises from about 0.2 mM to about 2 mM fucose. In certain embodiments, the culture media comprises from about 0.25 mM to about 1 mM fucose. In certain embodiments, the culture media comprises about 0.6 mM fucose. In certain embodiments, the culture media comprises about 0.5 mM fucose. In certain embodiments, the culture media comprises about 0.4 mM fucose. In certain embodiments, the culture media comprises about 0.45 mM fucose. In certain embodiments, the culture media comprises about 0.55 mM fucose. Patent Application

[0232] CLIMB -009 / 01 WO 40193 / 34

[0233] The table below provides the sequences for nucleic acids, proteins, and peptides discussed herein. Patent Application

[0234] CLIMB -009 / 01 WO 40193 / 34 Patent Application

[0235] CLIMB -009 / 01 WO 40193 / 34 Patent Application

[0236] CLIMB -009 / 01 WO 40193 / 34 Patent Application

[0237] CLIMB -009 / 01 WO 40193 / 34

[0238] Incorporation by Reference

[0239] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, publicly accessible databases, have been made Patent Application CLIMB -009 / 01 WO 40193 / 34 throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0240] Equivalents

[0241] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Patent ApplicationCLIMB -009 / 01 WO 40193 / 34CLAIMS1. A process for preparation of an anti -CD 19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

2. The process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metl 1, Vai 19, Ser51, and Leu53; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

3. The process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or more residues corresponding to IlelO, Metl 1, Vall9, Ser51, and Leu53; and the process comprises production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

4. The process of claims 1-3, wherein the anti -CD 19 antibody variable domain comprises a heavy chain variable region of SEQ ID NO: 17 and / or a light chain variable region of SEQ ID NO: 29.

5. The process of claims 1-4, wherein the CHO cells are CHO DG44 cells.

6. The process of claims 1-4, wherein the process does not comprise the use of live viruses and / or infectious retroviral particles.Patent Application CLIMB -009 / 01 WO 40193 / 347. The process of claims 1 -4, wherein the process comprises one or more of the below steps: thawing the CHO cells; culturing the thawed CHO cells; harvest the cultured CHO cells; isolate the anti-CD19 antibody; and purify the anti-CD19 antibody.

8. The process of claim 1, wherein the heavy chain variable region having, compared to SEQ ID NO: 13, one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

9. The process of claim 1, wherein the heavy chain variable region is SEQ ID NO: 13 comprising an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93.

10. The process of claim 1, wherein the heavy chain variable region has one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

11. The process of claim 1, wherein the heavy chain variable region is the amino acid sequence of SEQ ID NO: 17.

12. The process of claim 1, wherein the anti-CD19 antibody variable domain further comprises a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metl l, Vai 19, Ser51, and Leu53.

13. The process of claim 1, wherein the light chain variable region is at least 95% identical to SEQ ID NO: 25.

14. The process of claim 2, wherein the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, Metl ILeu, Vall9Ala, Ser51Asp, and Leu53Thr.

15. The process of claim 2, wherein the light chain variable region is SEQ ID NO: 25 comprising an amino acid substitution at one or more residues corresponding to IlelO, Metl l, Vai 19, Ser51, and Leu53.Patent ApplicationCLIMB -009 / 01 WO 40193 / 3416. The process of claim 2, wherein the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, Metl ILeu, Vall9Ala, Ser51Asp, and Leu53Thr.

17. The process of claim 2, wherein the light chain variable region is the amino acid sequence of SEQ ID NO: 29.

18. The process of claim 3, wherein the heavy chain variable region comprises one or more of substitutions Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

19. The process of claim 3, wherein the light chain variable region comprises one or more of substitutions IlelOThr, Metl ILeu, Vall9Ala, Ser51Asp, and Leu53Thr.

20. The process of claim 1, wherein the heavy chain variable region comprises SEQ ID NO: 17 and the light chain variable region comprises SEQ ID NO: 29.

21. A method for treatment of a disease by administration of anti-CD19 antibody, wherein the anti-CD19 antibody comprises variable domain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93; and the anti -CD 19 antibody is prepared by a process comprising production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

22. A method for treatment of a disease by administration of an anti-CD19 antibody, wherein the anti-CD19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metl l, Vai 19, Ser51, and Leu53; and the anti -CD 19 antibody is prepared by a process comprising production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

23. A method for treatment of a disease by administration of an anti-CD19 antibody, wherein the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53; andPatent ApplicationCLIMB -009 / 01 WO 40193 / 34 the anti-CD19 antibody is prepared by a process comprising production of the anti-CD19 antibody in Chinese Hamster Ovary (CHO) cells.

24. The method of claims 21-23, wherein the anti-CD19 antibody variable domain comprises a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 29.

25. The method of claims 21-24, wherein the CHO cells are CHO DG44 cells.

26. The method of claims 21-24, wherein the process does not comprise the use of live viruses and / or infectious retroviral particles.

27. The method of claims 21-24, wherein the process comprises one or more of the below steps: thawing the CHO cells; culturing the thawed CHO cells; harvest the cultured CHO cells; isolate the anti-CD19 antibody; and purify the anti-CD19 antibody.

28. The method of claim 21, wherein the heavy chain variable region having, compared to SEQ ID NO: 13, one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

29. The method of claim 21, wherein the heavy chain variable region is SEQ ID NO: 13 comprising an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93.

30. The method of claim 21, wherein the heavy chain variable region has one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.Patent ApplicationCLIMB -009 / 01 WO 40193 / 3431 . The method of claim 21 , wherein the heavy chain variable region is the amino acid sequence of SEQ ID NO: 17.

32. The method of claim 21, wherein the anti-CD19 antibody further comprises a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53.

33. The method of claim 21, wherein the light chain variable region is at least 95% identical to SEQ ID NO: 25.

34. The method of claim 22, wherein the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, Metl lLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

35. The method of claim 22, wherein the light chain variable region is SEQ ID NO: 25 comprising an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53.

36. The method of claim 22, wherein the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, MetllLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

37. The method of claim 22, wherein the light chain variable region is the amino acid sequence of SEQ ID NO: 29.

38. The method of claim 23, wherein the heavy chain variable region comprises one or more of substitutions Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

39. The method of claim 23, wherein the light chain variable region comprises one or more of substitutions IlelOThr, Metl lLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

40. The method of claims 21-24, wherein the disease is cancer or an autoimmune disorder, and preferably said autoimmune disorder is primary membranous nephropathy (PMN).Patent ApplicationCLIMB -009 / 01 WO 40193 / 3441 . A process for preparation of an anti-CD19 antibody, wherein: the anti -CD 19 antibody comprises variable domain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93; and the process comprises production of the anti-CD19 in cells, and fucosylation of the anti-CD19 antibody.

42. A process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vai 19, Ser51, and Leu53; and the process comprises production of the anti-CD19 in cells, and fucosylation of the anti-CD19 antibody.

43. A process for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53; and the process comprises production of the anti-CD19 in cells, and fucosylation of the anti-CD19 antibody.

44. The process of claims 41-43, wherein the anti-CD19 antibody variable domain comprises a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 29.

45. The process of claims 41-44, wherein the cells are Chinese Hamster Ovary (CHO) cells.

46. The process of claim 45, wherein the CHO cells are CHO DG44 cells.Patent ApplicationCLIMB -009 / 01 WO 40193 / 3447. The process of claim 45, wherein the process does not comprise the use of live viruses and / or infectious retroviral particles.

48. The process of claim 45, wherein the process comprises one or more of the below steps: thawing the CHO cells; culturing the thawed CHO cells; isolate the anti-CD19 antibody; and purify the anti-CD19 antibody.

49. The process of claims 41-44, wherein CHO cells are grown in a media comprising fucose.

50. The process of claim 49, wherein quantity of fucose in the media is modulated to achieve optimal level of fucosylation on the anti-CD19 antibody.

51. The process of claim 50, wherein the media comprises from about 0.2 mM to about 2 mM fucose52. The process of claim 50, wherein the media comprises from about 0.1 mM to about 2.5 mM fucose.

53. The process of claim 50, wherein the media comprises from about 0.2 mM to about 1.5 mM fucose.

54. The process of claim 50, wherein the media comprises from about 0.25 mM to about 1 mM fucose.

55. The process of claim 50, wherein the media comprises about 0.5 mM fucose.

56. The process of claim 50, wherein the media comprises about 0.4 mM fucose.

57. The process of claim 50, wherein the media comprises about 0.6 mM fucose.

58. The process of claim 50, wherein the media does not include fucose.

59. The process of claim 50, wherein the cells are cultured from about 5 days to about 30 days.Patent ApplicationCLIMB -009 / 01 WO 40193 / 3460. The process of claim 50, wherein the cells are cultured from about 10 days to about 25 days.

61. The process of claim 50, wherein the cells are cultured from about 12 days to about 20 days.

62. The process of claim 50, wherein the cells are cultured from about 12 days to about 15 days.

63. The process of claim 50, wherein the anti-CD19 antibody is 30% to about 75% fucosylated.

64. The process of claim 50, wherein the anti -CD 19 antibody is 35% to about 70% fucosylated.

65. The process of claim 50, wherein the anti-CD19 antibody is about 40% fucosylated.

66. The process of claim 50, wherein the anti -CD 19 antibody is about 45% or 46% fucosylated.

67. The process of claim 50, wherein the anti -CD 19 antibody is about 50% fucosylated.

68. The process of claim 50, wherein the anti-CD19 antibody is about 55% fucosylated.

69. The process of claim 50, wherein the anti -CD 19 antibody is about 60% fucosylated.

70. The process of claim 50, wherein the anti -CD 19 antibody is about 65% fucosylated.

71. The process of claim 50, wherein the anti-CD19 antibody has less than 2% fucosylation.

72. The process of claim 41, wherein the heavy chain variable region having, compared to SEQ ID NO: 13, one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

73. The process of claim 41, wherein the heavy chain variable region is SEQ ID NO: 13 comprising an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93.Patent Application CLIMB -009 / 01 WO 40193 / 3474. The process of claim 41, wherein the heavy chain variable region has one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

75. The process of claim 41, wherein the heavy chain variable region is the amino acid sequence of SEQ ID NO: 17.

76. The process of claim 41, wherein the anti-CD19 antibody further comprises a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53.

77. The process of claim 41, wherein the light chain variable region is at least 95% identical to SEQ ID NO: 25.

78. The process of claim 42, wherein the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, Metl lLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

79. The process of claim 42, wherein the light chain variable region is SEQ ID NO: 25 comprising an amino acid substitution at one or more residues corresponding to IlelO, Metll, Vall9, Ser51, and Leu53.

80. The process of claim 42, wherein the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, MetllLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

81. The process of claim 42, wherein the light chain variable region is the amino acid sequence of SEQ ID NO: 29.

82. The process of claim 43, wherein the heavy chain variable region comprises one or more of substitutions Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

83. The process of claim 43, wherein the light chain variable region comprises one or more of substitutions IlelOThr, MetllLeu, Vall9Ala, Ser51Asp, and Leu53Thr.Patent ApplicationCLIMB -009 / 01 WO 40193 / 3484. The process of claim 49, wherein volume of media is at least 200L.

85. The process of claim 49, wherein volume of media is about 250L.

86. The process of claim 49, wherein volume of media is at least 500L.

87. The process of claim 49, wherein volume of media is about 500L.

88. The process of claim 49, wherein volume of media is at least 1000L.

89. The process of claim 49, wherein volume of media is about OOL.

90. The process of claim 49, wherein volume of media is 200L, 500L, or lOOOL.

91. A composition for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a heavy chain variable region that is at least 90% identical to SEQ ID NO: 13 and has an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and the anti-CD19 antibody is fucosylated; and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose.

92. A composition for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises variable domain comprising a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metl l, Vai 19, Ser51, and Leu53; and the antiCD 19 antibody is fucosylated; and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose.

93. A composition for preparation of an anti-CD19 antibody, wherein: the anti-CD19 antibody comprises SEQ ID NO: 13 with an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93, and SEQ ID NO: 25 with an amino acid substitution at one or morePatent ApplicationCLIMB -009 / 01 WO 40193 / 34 residues corresponding to II e 10, Metl 1 , Vai 19, Ser51 , and Leu53, and Leu53; and the antiCD 19 antibody is fucosylated; and the composition comprises a cell growth media for Chinese Hamster Ovary (CHO) cells and fucose.

94. The composition of claims 91-93, wherein the anti-CD19 antibody variable domain comprises a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 29.

95. The composition of claims 91-94, wherein the CHO cells are CHO DG44 cells.

96. The composition of claim 95, wherein the process does not comprise the use of live viruses and / or infectious retroviral particles.

97. The composition of claim 95, wherein the process comprises one or more of the below steps: thawing the CHO cells; culturing the thawed CHO cells; isolate the anti-CD19 antibody; and purify the anti-CD19 antibody.

98. The composition of claims 91-94, wherein CHO cells are grown in a media comprising fucose.

99. The composition of claims 91-94, wherein the media comprises from about 0.1 mM to about 2.5 mM fucose.

100. The composition of claims 91-94, wherein the media comprises from about 0.2 mM to about 2 mM fucose.

101. The composition of claims 91-94, wherein the media comprises from about 0.2 mM to about 1.5 mM fucose.

102. The composition of claims 91-94, wherein the media comprises from about 0.25 mM to about 1 mM fucose.Patent ApplicationCLIMB -009 / 01 WO 40193 / 34103. The composition of claims 91-94, wherein the media comprises about 0.5 mM fucose.

104. The composition of claims 91-94, wherein the media comprises about 0.4 mM fucose.

105. The composition of claims 91-94, wherein the media comprises about 0.6 mM fucose.

106. The composition of claims 91-94, wherein the cells are cultured from about 5 days to about 30 days.

107. The composition of claims 91-94, wherein the cells are cultured from about 10 days to about 25 days.

108. The composition of claims 91-94, wherein the cells are cultured from about 12 days to about 20 days.

109. The composition of claims 91-94, wherein the cells are cultured from about 12 days to about 15 days110. The composition of claim 91, wherein the heavy chain variable region having, compared to SEQ ID NO: 13, one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

111. The composition of claim 91, wherein the heavy chain variable region is SEQ ID NO: 13 comprising an amino acid substitution at one or more residues corresponding to Gln5, Argl9, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93.

112. The composition of claim 91, wherein the heavy chain variable region has one or more amino acid substitutions selected from the group consisting of Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

113. The composition of claim 91, wherein the heavy chain variable region is the amino acid sequence of SEQ ID NO: 17.Patent Application CLIMB -009 / 01 WO 40193 / 34114. The composition of claim 91 , wherein the anti-CD19 antibody further comprises a light chain variable region that is at least 90% identical to SEQ ID NO: 25 and has an amino acid substitution at one or more residues corresponding to IlelO, Metl 1, Vall9, Ser51, and Leu53.

115. The composition of claim 91, wherein the light chain variable region is at least 95% identical to SEQ ID NO: 25.

116. The composition of claim 92, wherein the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, MetllLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

117. The composition of claim 92, wherein the light chain variable region is SEQ ID NO: 25 comprising an amino acid substitution at one or more residues corresponding to IlelO, Metl l, Vai 19, Ser51, and Leu53.

118. The composition of claim 92, wherein the light chain variable region has one or more amino acid substitutions selected from the group consisting of IlelOThr, MetllLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

119. The composition of claim 92, wherein the light chain variable region is the amino acid sequence of SEQ ID NO: 29.

120. The composition of claim 93, wherein the heavy chain variable region comprises one or more of substitutions Gln5Glu, Argl9Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp, Ser88Ala, and Val93Thr.

121. The composition of claim 93, wherein the light chain variable region comprises one or more of substitutions IlelOThr, Metl lLeu, Vall9Ala, Ser51Asp, and Leu53Thr.

122. The composition of any of claims 91-93, wherein the quantity of fucose in the media is a pre-determined amount to achieve optimal level of fucosylation on the antiCD 19 antibody.

123. The composition of any of claims 122, wherein the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is 30% to about 75% fucosylated.Patent ApplicationCLIMB -009 / 01 WO 40193 / 34124. The composition of any of claims 122, wherein the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is 35% to about 70% fucosylated.

125. The composition of any of claims 122, wherein the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is about 40% fucosylated.

126. The composition of any of claims 122, wherein the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is about 45% or 46% fucosylated.

127. The composition of any of claims 122, wherein the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is about 50% fucosylated.

128. The composition of any of claims 122, wherein the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is 55% fucosylated.

129. The composition of any of claims 122, wherein the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is 60% fucosylated.

130. The composition of any of claims 122, wherein the pre-determined amount of fucose in the media is a pre-determined amount that results in the anti-CD19 antibody is 65% fucosylated.

131. The composition of claim 98, wherein volume of the composition is at least 200L.

132. The composition of claim 98, wherein volume of media is about 250L.

133. The composition of claim 98, wherein volume of media is at least 500L.

134. The composition of claim 98, wherein volume of media is about 500L.

135. The composition of claim 98, wherein volume of media is at least 1000L.

136. The composition of claim 98, wherein volume of media is about 1000L.

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